This invention is directed toward a method for obtaining nucleic acid ligands against target proteins without directly purifying the target proteins. The method used in the invention is called the SELEX process, which is an acronym for Systematic Evolution of Ligands by EXponential enrichment.
The past ten years have seen phenomenal advances in the characterization of the genomes of many species. Indeed, the human genome sequencexe2x80x94encoding for approximately 100,000 proteinsxe2x80x94is now substantially complete. With the completion of a genome sequence, the linear amino acid sequences of all the proteins potentially encoded by that genome are known. The goal of the biomedical research community is to use the genomic data to learn about the functions of the proteins that are encoded by the genome, and then determine the role that these proteins play in pathogenesis and disease. Unfortunately, the tools for identifying the function of proteinsxe2x80x94their structural or enzymatic activities, and their level of synthesisxe2x80x94are dramatically less well developed than those for determining genomic sequences. As a result, the characterization of the functions of such proteins is the rate limiting step in the exploitation of genomic data for the development of new diagnostic and therapeutic agents.
Although some proteins are identified solely through the existence of their coding sequence in the genome, more functional approaches to protein identification and characterization have been devised. For example, one approach involves isolating all the proteins that are expressed under predetermined conditions in a certain tissue, then resolving those proteins from one another by electrophoresis on a 2-dimensional gel. Following separation, individual protein xe2x80x9cspotsxe2x80x9d on the gel are picked and proteolytically-digested to yield peptides. The resulting peptides can be analyzed by reiterative mass spectrometry in order to determine their (partial) linear amino acid sequences. Finally, the amino acid sequences of the peptides are used to search genomic or cDNA sequences in order to obtain the DNA sequence that encodes the protein from which the peptide was derived. In this way, it is possible to prepare protein and gene expression profiles. However, because this approach is extremely labor and capital-intensivexe2x80x94requiring several days to analyze a single gelxe2x80x94it is not suited to high-throughput, routine diagnostic applications.
Regardless of the manner in which a protein implicated in disease is initially identified, it is ultimately crucial to obtain ligands to that protein, because such ligands can serve as therapeutic or diagnostic reagents. In order to generate ligands, it is necessary to have a purified source of the protein. However, because important proteins are often present in vanishingly-small amounts in biological tissues, purificationxe2x80x94if it is even possible at allxe2x80x94is often a costly, labor-intensive, and time-consuming procedure. Expression of proteins is also fraught will difficulties, often because of the complexity of the post-translational modifications seen in mammalian proteins. Because of these difficulties, there is a need in the field of functional genomics for a method of generating ligands of target proteins without first requiring that the target protein be directly purified.
There have been several attempts in the art to overcome these difficulties by generating ligands of synthetic peptides with the same linear amino acid sequence as a portion of the target protein. The hope in this approach is that the ligandxe2x80x94typically an antibodyxe2x80x94to the peptide will recognize the same peptide in the natural context of the intact protein. There are two fundamental problems with this approach. First, because protein structures have a large internal mass compared to their external surface, most peptide sequences from a specific protein lie within the internal mass of the protein and are not exposed to solvent. As a result, many ligands to peptides will not be able to access the same peptides within the intact protein. Second, isolated peptides typically have random, undefined structures, whereas the same peptide in the intact protein will have one or a few defined structures as a result of intra-molecular constraints imposed upon it. Because ligands are generated using the isolated peptide as the target, many ligands will not recognize the defined peptide structure within the intact protein. Both of these problems cause anti-peptide antibodies to have weak affinities for the proteins that contain the same peptides.
A new class of non-protein-based ligands is found in nucleic acid molecules. The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment,xe2x80x9d now abandoned, U.S. Pat. No. 5,475,096 entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d each of which is specifically incorporated by reference herein. Each of these patents and applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, and U.S. patent application Ser. No. 08/443,959 filed May 18, 1995, both entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d and both now abandoned, and U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, U.S. patent application Ser. No. 09/459,553, filed Dec. 13, 1999, and U.S. patent application Ser. No. 09/619,213, filed Jul. 17, 2000, each of which is entitled xe2x80x9cSystematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,xe2x80x9d all describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. These patents and patent applications are referred to in this application collectively as xe2x80x9cthe photo SELEX process applications.xe2x80x9d
U.S. Pat. No. 5,580,737 entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938 entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Rev,xe2x80x9d describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. Pat. No. 5,705,337 entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX,xe2x80x9d describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985 entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of 2xe2x80x2 Modified Pyrimidine Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,xe2x80x9d and U.S. Pat. No. 5,683,867 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,xe2x80x9d respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. Pat. No. 6,011,020 entitled xe2x80x9cNucleic Acid Complexesxe2x80x9d.
The SELEX process has been adapted in order to allow the high-throughput, automated generation of high affinity nucleic acid ligands to targets of interest. Methods and apparatus for automated generation of nucleic acid ligands are described in U.S. patent application Ser. No. 09/232,946, filed Jan. 19, 1999, U.S. patent application Ser. No. 09/356,233 filed Jul. 16, 1999, and U.S. patent application Ser. No. 09/616,284, filed Jul. 14, 2000, each of which is entitled xe2x80x9cMethods and Apparatus for the Automated Generation of Nucleic Acid Ligands.xe2x80x9d We refer to these patent applications collectively as xe2x80x9cthe automated SELEX process applications.xe2x80x9d
Nucleic acid ligands may be attached to the surface of solid supports to form microarrays. Such microarrays (also commonly referred to as xe2x80x9cbiochipsxe2x80x9d), and methods for their manufacture and use, are described in U.S. patent application Ser. No. 08/990,436, filed Dec. 15, 1997, U.S. patent application Ser. No. 08/211,680, filed Dec. 14, 1998, now abandoned, Patent Cooperation Treaty Application Serial No. PCT/US98/26515, filed Dec. 14, 1998, U.S. patent application Ser. No. 09/581,465, filed Jun. 12, 2000, each of which is entitled xe2x80x9cNucleic Acid Ligand Diagnostic Biochip.xe2x80x9d We refer to these patent applications collectively as xe2x80x9cthe biochip applications.xe2x80x9d
One potential problem encountered in the diagnostic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 9, 1993, now abandoned, and U.S. Pat. No. 5,660,985, both entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotidesxe2x80x9d, and U.S. patent application Ser. No. 09/362,578 filed Jul. 28, 1999, entitled xe2x80x9cTranscription-free SELEXxe2x80x9d, each of which is specifically incorporated herein by reference. Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2xe2x80x2-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3xe2x80x2 and 5xe2x80x2 modifications such as capping. In preferred embodiments of the instant invention, the nucleic acid ligands are DNA molecules that are modified with a photoreactive group on 5-position of pyrimidine residues. The modifications can be pre- or post-SELEX process modifications.
Each of the above described patent applications, many of which describe modifications of the basic SELEX procedure, are specifically incorporated by reference herein in their entirety.
There are a number of prior art teachings of nucleic acid ligands to unconstrained peptides. For example, Nieuwlandt et al, Biochemistry 34: 5651-5659 (1995) describe a high-affinity (190 nm Kd) nucleic acid ligand to the 11 amino acid tachykinin substance P. Ellington and Xu, Proc. Natl. Acad. Sci. USA, 93: 7475-7480 (1996), teach that a nucleic acid ligand to a 17-mer peptide fragment of Human Immunodeficiency Virus (HIV) Rev protein can bind specifically to the same peptide within intact Rev protein. However, because of the aforementioned problems, the affinity of the nucleic acid ligand for the isolated 17-mer peptide is significantly better than for the intact Rev protein i.e., the Kd for the peptide is lower than the Kd for the intact protein.
The present invention provides for the first time a method for obtaining nucleic acid ligands that bind to target proteins without requiring a source of purified target protein.
The methods provided herein use the SELEX process for ligand generation. In particular, the methods of the instant invention allow the generation of nucleic acid ligands to protein targets that are not generally available in purified form, but for which a least a partial cDNA or genomic sequence is known. The nucleic acid ligands of the instant invention are initially generated by the SELEX process, using, as SELEX targets, peptides corresponding in sequence to the target protein, or derivatives of target proteins (including fragments of target proteins) expressed in vitro or in vivo. This method generates candidate nucleic acid mixtures that are enriched for nucleic acid ligands with affinity to the peptide or expressed protein. Further enrichment of the candidate mixture for those nucleic acid ligands that also have affinity for the intact, native target protein may optionally be achieved by performing an additional number of rounds of the SELEX process using, as a SELEX target, a complex mixture suspected of containing the target protein e.g., a tissue extract or biological fluid. Although such complex mixtures may contain many other proteins, and may contain only minute quantities of the target protein, the initial enrichment performed using the peptide or expressed protein as a SELEX target nevertheless allows high affinity nucleic acid ligands of the target protein to be obtained.
Nucleic acid ligands generated according to the methods of the instant invention will have a great utility as diagnostic and prognostic reagents, as novel therapeutics, and as agents for the identification of novel therapeutic targets.